Solid-phase purification of synthetic nucleic acid sequences

A method of purifying an oligonucleotide or an oligonucleotide analog composed of “b” nucleotides from a mixture comprising the oligonucleotide or oligonucleotide analog and at least one oligonucleotide or oligonucleotide analog composed of “a” nucleotides, wherein b≠a, the method comprising:

• (i) providing a protected nucleoside or nucleoside analog of formula (I) or (Ia) functionalized with an activatable phosphorus-containing entity
• (ii) providing a mixture comprising an optionally protected first oligonucleotide or oligonucleotide analog V′ composed of b-1 nucleotides or nucleotide analogs and having a free 5′-terminal OH group, wherein the first oligonucleotide V′ comprises phosphate or phosphorothioate triester linkages, or a combination thereof, and wherein the first oligonucleotide V′ is linked at its 3′-terminus to a solid support, wherein at least one oligonucleotide or oligonucleotide analog composed of “a” nucleotides is also linked to the solid support;
• (iii) coupling the first oligonucleotide V′ with the protected nucleoside or nucleoside analog of formula (I) or (Ia), to provide a second oligonucleotide of the formula (II) or (IIa)
• (iv) oxidizing or sulfurizing, optionally deprotecting, and cleaving the second oligonucleotide or oligonucleotide analog of the formula (II) or (IIa) from the solid support to form a mixture comprising a third oligonucleotide of the formula (III) or (IIIa)
• wherein V is the moiety resulting after optional deprotection of the second oligonucleotide or oligonucleotide analog and wherein V is not linked to the solid support;
• (v) reacting the mixture comprising the third oligonucleotide or oligonucleotide analog of the formula (III) or (Ma) with a silica-attached linker compound of the formula:
• wherein A, K, and L are independently C2-C10 alkanediyl and R6 is H or C1-C6 linear or branched alkyl or C3-C8 cycloalkyl, and wherein
• is silica, to form a linker-attached oligonucleotide or oligonucleotide analog of the formula (IV) or (IVa)
• (vi) washing the linker-attached oligonucleotide of the formula (IV) or (IVa) with at least one solvent or a mixture of solvents to remove the oligonucleotide(s) or oligonucleotide analog(s) composed of “a” nucleotides;
• (vii) treating the linker-attached oligonucleotide or oligonucleotide analog of formula (IV) or (IVa) with a desilylation agent;
• and
• (viii) isolating the purified oligonucleotide or oligonucleotide analog composed of “b” nucleotides from the product of step (vii);
• wherein
• B is an optionally protected nucleobase or an optionally protected nucleobase analog;
• D and E are independently C2-C10 alkanediyl;
• n is 1 to 4;
• R1 is C1-C6 linear or branched alkyl or C3-C8 cycloalkyl;
• R2 is hydrogen or linear or branched C1-C6 alkyl;
• R3 is linear or branched C3-C6 alkyl;
• J is H or OR7 wherein R7 is a reversible or permanent hydroxyl protecting group;
• X is O or S;
• Y is H or C1-C6 linear alkyl;
• W is a lone pair of electrons or an oxo (═O) function;
• when W is a lone pair of electrons, Z is NR4R5 wherein R4 and R5 are individually C1-C6 alkyl, C6-C10 arylated C1-C6 alkyl, or C3-C8 cycloalkyl or R4 and R5 taken together with the nitrogen atom to which they are attached form a saturated 3-10 membered heterocyclic ring optionally including one or more additional heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and Q is OT wherein T is a reversible or permanent hydroxyl protecting group;
• and
• when W is an oxo function, Z is H and Q is O−.